1 Feeding preferences of the 2 setosum (Leske, 1778) in Taklong Island National 3 Marine Reserve, Guimaras, Philippines 4 Jennelle Christianne S. Luza1, Maria Celia D. Malay1, 5 1 Division of Biological Sciences, College of Arts and Sciences, University of the Philippines 6 Visayas, Miagao, Iloilo, Philippines 7 8 Corresponding Author: 9 Jennelle Christianne Luza1 10 Brgy. Inzo Arnaldo Village, Roxas City, Capiz, 5800, Philippines 11 Email address: [email protected] 12 13 14 Abstract 15 Background. Sea urchins are keystone herbivores that greatly influence primary productivity, 16 algal abundance and scleractinian recruitment. The long-spined black sea urchin Diadema 17 setosum is widespread and abundant in reef flats throughout the Philippines. Prior studies 18 regarding the feeding preference of D. setosum have been conducted overseas, but little is known 19 about the impact of the echinoid herbivory on reef flat communities in the Philippines. Feeding 20 preferences of D. setosum on four common marine plant species, Halimeda macroloba, 21 Ceratodictyon spongiosum, Padina sp., and acoroides were investigated at the 22 University of the Philippines Visayas Marine Biological Laboratory, located in Taklong Island 23 National Marine Reserve (TINMR), Guimaras. 24 Methods. Two food choice experiments were conducted; choice feeding and no-choice feeding. 25 The outcome of choice feeding experiments, expressed as consumption (in g) and percent 26 consumption (%), were used to determine its feeding preferences. The two most preferred feeds 27 determined were then used in no-choice feeding experiment to measure its consumption rate 28 (g⸱echinoid-1⸱hr-1). 29 Results. Results of the choice feeding experiment show that D. setosum significantly prefers C. 30 spongiosum (4.83 ± 2.56 g consumption or 32.2%) and H. macroloba (3.73 ± 2.27 g or 24.8%), 31 and avoids E. acoroides (only 0.17 ± 0.22 g or 1.13%) (F= 5.423, p < 0.05). The no-choice 32 feeding experiment between preferred feeds show H. macroloba was consumed more (0.22 ± 33 0.16 g⸱echinoid-1⸱hr-1) than C. spongiosum (0.15 ± 0.05 g⸱echinoid-1⸱hr-1) although there was no 34 significant difference (p > 0.05) in consumption rate. Results of the no-choice feeding 35 experiment may have been affected by poor water quality and are considered inconclusive. 36 Nevertheless, the study supports the ecological role of D. setosum as an important herbivore that 37 regulates certain macroalgal species in TINMR through its grazing activities. 38 39 Introduction 40 Sea urchins play a vital role in marine ecosystems especially in shallow tropical seas. They are 41 considered keystone herbivores as they effectively influence marine plant populations such as 42 and , their primary productivity and abundance, and scleractinian coral 43 recruitment by grazing on algae that compete with (Shunula & Ndibalema, 1986; Alves et

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27733v1 | CC BY 4.0 Open Access | rec: 15 May 2019, publ: 15 May 2019 44 al., 2003). Studies about sea urchins peaked after a massive mortality event of Diadema 45 antillarum in the Caribbean in 1983 which caused drastic changes in the community 46 structure (Lessios et al., 1984). The disappearance of urchins in the area resulted in increased 47 density and diversity of algal species and led to higher algal cover (Solandt & Campbell, 2001). 48 In terms of feeding ecology, grazing urchins operate as either generalists or specialists in 49 a community (Stimson, Cunha & Philippoff, 2007). Some urchins feed on available algae in its 50 environment (Solandt & Campbell, 2001). In Hawaii, studies about the native sea urchin 51 Tripneustes gratilla showed that it can function as biocontrol agent for invasive algae (Stimson, 52 Cunha & Philippoff, 2007; Westbrook et al., 2015). However, Tomas, Box & Terrados et al. 53 (2011) suggested that some sea urchin species like Paracentrotus lividus do not function as 54 control agent of invasive algal species. Other studies report that different species of sea urchins 55 exhibit feeding preferences (Larson, Vadas & Keser, 1980; Hay, Lee & Guieb, 1986; Solandt & 56 Campbell, 2001; Tuya et al., 2001; Stimson, Cunha & Philippoff, 2007; Kasim, 2009; Lyimo et 57 al., 2011; Seymour et al., 2013). 58 Food preferences of sea urchins may be influenced by the distribution and abundance of 59 its food source (Seymour et al., 2013), the chemical and morphological properties (Shunula & 60 Ndibalema, 1986; Solandt & Campbell, 2001; Tuya et al., 2001; Erickson et al., 2006; Souza, de 61 Olivera & Pereira, 2008; Seymour et al., 2013), and caloric content (Larson, Vadas & Keser, 62 1980) of plant species, and the different stages of sea urchin development (Westbrook et al., 63 2015). Hay, Lee & Guieb (1986) also stated that chemotaxis correlated with daytime and 64 nighttime hours affect the feeding behavior of the urchin. Additionally, the preferred and non- 65 preferred feeds of sea urchin differ at different seasons of the year (Larson, Vadas & Keser, 66 1980; Seymour et al., 2013). 67 , a black and long-spined sea urchin having distinct white dots on its 68 body, is widespread along Indo-Pacific regions including Philippines and is thought to be 69 ecologically important in shallow subtidal ecosystems. Their gonads serve as a delicacy in many 70 local communities and are targeted as wild fishery. Diadema setosum forages at night in the 71 tropics to avoid predators (Lawrence & Hughes-Games, 1972). Studies have reported feeding 72 preferences of D. setosum on a specific macroalgal species vary in different areas around the 73 world (Shunula & Ndibalema, 1986; Moore et al., 2019). Tatsuya, Miyuki & Akira (2016), also 74 reported that grazing and high densities of D. setosum control algal coverage and density on the 75 seaweed bed ecosystems along the central coast of Japan. However, in Singapore reefs, D. 76 setosum is not an important component of the herbivore guild (Goh & Lim, 2015). Seasonal 77 changes have also been reported in the size of the gut of some sea urchins related to changes in 78 food availability (Lawrence, Lawrence & Watts, 2013), there were no changes in D. setosum in 79 or on Kenyan reefs (Pearse, 1974; Muthiga, 2003). Diadema setosum was also found 80 out to be a key symbiont of cardinalfish Pterapogon kauderni in Indonesia (Moore et al., 2019). 81 Interestingly, the work of Coppard & Campbell (2007) in Fiji show that sea urchins under the 82 same , Diadema setosum and D. savignyi exhibit selective grazing, with distinct feeding 83 preferences. 84 While many studies about the feeding ecology of D. setosum have been conducted in 85 different countries, very little research has been undertaken in Southeast Asia especially in the 86 Philippines. 87 The study was conducted to determine if D. setosum exhibits a preference for different 88

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27733v1 | CC BY 4.0 Open Access | rec: 15 May 2019, publ: 15 May 2019 89 feeds ( and macroalgae species) presently dominating in Taklong Island National Marine 90 Reserve (TINMR), Guimaras. The study also determined the rate of consumption of D. setosum 91 for different feeds. Feeding preference assay was done by (a) determining the consumption (g) of 92 the urchins when offered a choice of feeds, to see what the sea urchin preferred, then present the 93 values in percent consumption (%), and (b) investigating how much of the preferred feeds can D. 94 setosum consume in a given amount of time (consumption rate) using single diet experiment. 95 Since sea urchins mainly feed on micro- and macroalge, and others on seagrasses, detrital 96 particles and corals (Cabanillas-Teran et al., 2016), these grazing herbivores can be implicated as 97 a driver of phase shifts in marine environments (Kriegsch et al., 2016). Ecologists are greatly 98 interested on the feeding preferences of sea urchins as it not only determines the phase shifts in 99 an ecosystem, such as shifting from a coral-dominated to macroalgal-dominated system, but can 100 also provide trophic links in community food webs. Aquaculturists also use feeding preferences 101 to determine the quantity and quality of food ingested to determine the optimal physiological 102 condition of sea urchins. Understanding the feeding preference and feeding rate of D. setosum 103 will help predict the impact of herbivory on coral and seagrass communities since feeding 104 preferences interact with plant competitive abilities, life histories, and physical tolerances in 105 determining the impact of a grazer on the marine benthic community (Coppard & Campbell, 106 2007). This study will also help in sustainable management of both the sea urchin and marine 107 plant species in an ecosystem. 108 109 Materials & Methods 110 Experimental organisms 111 Twenty (20) Diadema setosum sea urchins were collected from the rocky shore areas in the UPV 112 Channel separating Taklong Island from mainland Guimaras, located within the Taklong Island 113 National Marine Reserve (TINMR), Guimaras. Utmost care was taken in collecting D. setosum 114 since its long, black spines are fragile. A custom-made sea urchin scooper made from thin rebar 115 was designed for properly collecting the echinoids (Fig. 1). were then placed in large 116 bins (450 l) with aerated sea water, held just outside the laboratory. The sea urchins were starved 117 for at least 48 hours prior to use in assays to acclimatize and to overcome any possible period of 118 ingestive conditioning (Solandt & Campbell, 2001). The tanks were shaded from direct sunlight 119 and were exposed to natural photoperiod. 120

121 122 Figure 1. Custom-made tool for scooping sea urchins (design provided by Harilaos Lessios).

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27733v1 | CC BY 4.0 Open Access | rec: 15 May 2019, publ: 15 May 2019 123 124 A preliminary survey was done on the forereef of UPV Channel to determine the most 125 common marine plant species by snorkeling around the area. A plant species was considered 126 common if it was mostly seen throughout the survey area. The most common seagrass and 127 macroalgae species in the UPV Channel in TINMR were collected hours before the start of the 128 experiment. There were four experimental feeds namely Enhalus acoroides (seagrass), Padina 129 sp. (brown alga), Halimeda macroloba (green alga), and Ceratodictyon spongiosum (red 130 macroalgae symbiotic with sponge Haliclona cymaeformis) (Fig. 2). The identification of the 131 feeds was made in the laboratory using a field guide book of the seaweed species in the 132 Philippines (Trono, 1997) and identification cards of seagrass species found in the laboratory. 133

A B C D 134 135 Figure 2. Four different feeds used in feeding experiments; (A) Halimeda macroloba, (B) 136 Ceratodictyon spongiosum, (C) Enhalus acoroides, and (D) Padina sp. 137 138 Choice feeding experiment 139 The choice feeding experiment was conducted in January 11, 2019 with five (5) replicates to 140 observe differences in quantity eaten overnight. Methods were adapted from Solandt & 141 Campbell (2001), and Seymour & Dworjanyn (2013) with minor modifications. The urchins 142 used were 7-8 cm in test diameter. Three animals were placed in each large bin (450 l) with 143 aerated seawater exposed to natural light. Fifteen grams (15 g) of each feed type were wet 144 weighed using a top loading balance (water was removed prior to weighing by blotting with 145 paper towels), then divided into three clumps. Each of the three subdivided feeds was tied to a 146 stone using a rubber band to prevent the feed from floating and drifting away. The feeds were 147 placed randomly with at least 10 cm radial distance between each feed to prevent mix-up and so 148 that the urchins had an equal opportunity to graze on the feeds (Fig. 3, A and B). 149 The echinoids were introduced to the feeds at 17:30 hours and left to feed overnight. The 150 remaining feeds were removed from the bin after 16.5 hours, blotted dry, and weighed to 151 measure the quantity lost to grazing. A non-replicated control setup was run simultaneously with 152 the tests; the wet weight of feeds was manipulated to be the same weight as above and was 153 placed in a similar bin but without the sea urchins. The consumption of each feed (W) was 154 calculated using the formula from Seymour et al. (2013) which adjusts for any weight change in 155 the corresponding autogenic control (WC): 156 157 Consumption= 푊퐶푓푖푛푎푙 158 (푊푖푛푖푡푖푎푙 (푊퐶푖푛푖푡푖푎푙) − 푊푓푖푛푎푙)

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27733v1 | CC BY 4.0 Open Access | rec: 15 May 2019, publ: 15 May 2019 159 Finally, the percent consumption was calculated to provide insight into how much the echinoid 160 consumes: 161 162 Percent (%) consumption= 퐴푣푒푟푎푔푒 푐표푛푠푢푚푝푡푖표푛 163 푊푖푛푖푡푖푎푙 푥 100 164 No-choice feeding experiment 165 A no-choice feeding experiment was used to complement the choice feeding test by determining 166 the consumption rates of the feeds used. Preferred feeds were used to determine links between 167 preferences and consumption rates (Seymour et al., 2013). 168 Methods in no-choice feeding experiment were adapted from the work of Solandt & 169 Campbell (2001) with small alterations. The experiment was run in February 2, 2019 using the 170 same experimental containers as the choice feeding experiments. The urchins used were 7-8 cm 171 in test diameter. The feeds offered to the urchins were the two most preferred species during the 172 choice feeding experiment, namely Ceratodictyon spongiosum and Halimeda macroloba. Fifty 173 grams (50 g) of each preferred feed type was blotted dry, wet weighed using a top loading 174 balance, then divided into five clumps. Each of the three subdivided feeds was tied to a stone 175 using a rubber band to prevent the feed from floating and drifting away. Three replicates per feed 176 type were run in this experiment. One urchin was placed in each of the large bins with aerated 177 seawater. Only one feed type was introduced per bin (Fig. 3, C and D). 178 The feeds were introduced to the echinoids at 17:30 hrs and the experiments were left for 179 two days (48 hrs). The control consisted of the two preferred feeds (non-replicated) placed 180 together in large bin having the same water conditions as the treatments. After 48 hours, the 181 remaining feeds from the treatment and control bins were wet weighed. 182 Consumption rates of each feed type was calculated by dividing the consumption (g) with 183 duration of feeding experiment (in hours) and number of echinoids (1) per replicate (following 184 Seymour et al., 2013): 185 186 Consumption rate= 187 퐶표푛푠푢푚푝푡푖표푛 (푛표. 표푓 ℎ푟푠 푥 푒푐ℎ푖푛표푖푑)

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27733v1 | CC BY 4.0 Open Access | rec: 15 May 2019, publ: 15 May 2019 A B

C D 188 189 Figure 3. Experimental setups for the two feeding experiments; Choice feeding (A and B) and 190 No-choice feeding experiment (C and D). 191 192 Data analysis 193 Analyses of the statistical data were performed using the software package Statistical Package 194 for the Social Sciences (SPSS). 195 196 Choice feeding experiment 197 In the choice feeding preference experiment, Analysis of Variance (ANOVA) was used to 198 determine whether there was a significant difference in the consumption of the four feeds. Post- 199 hoc multiple comparisons (Tukey) test was also run to find paired differences in feeds 200 consumption. 201 202 No-choice feeding experiment 203 Comparison of the consumption rates (g·echinoid-1·hour-1) of the preferred feeds were 204 subsequently analyzed using non-parametric Mann-Whitney U test to determine whether there 205 was a significant difference in the consumption rates of the preferred feeds. 206 207 Results 208 Choice feeding experiment 209 The consumption of different feed types by D. setosum showed that Ceratodictyon spongiosum 210 was highly consumed (4.83 ± 2.56 g), followed closely by Halimeda macroloba (3.73 ± 2.27 g), 211 then Padina sp. (2.58 ± 1.73 g). Enhalus acoroides has the lowest consumption at only 0.17 ± 212 0.22 g (Table 1). 213

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27733v1 | CC BY 4.0 Open Access | rec: 15 May 2019, publ: 15 May 2019

Mean 95% Confidence Consum Standard Standard Interval for Mean N Minimum Maximum ption Deviation Error Lower Upper (g) Bound Bound Padina sp. 5 2.5760 1.73160 .77439 .4259 4.7261 .94 5.05 C. spongiosum 5 4.8340 2.55572 1.14295 1.6607 8.0073 1.33 7.33 H. macroloba 5 3.7320 2.26945 1.01493 .9141 6.5499 .07 5.67 E. acoroides 5 .1680 .21673 .09692 -.1011 .4371 -.06 .51 Total 20 2.8275 2.50065 .55916 1.6572 3.9978 -.06 7.33 214 Table 1. Descriptive statistics D. setosum food consumption in choice feeding experiments. 215 216 The minimum consumption of E. acoroides in one of the replicates was -0.06 g which 217 was an unusual result. It can be accounted to the systematic errors performed by the 218 experimenter during weighing and reweighing of the feeds. 219 Generally, the differences in consumption of the four feeds were significant (p = 0.009, 220 ANOVA; Appendix A). The echinoid significantly preferred C. spongiosum and H. macroloba 221 over the other two feeds (p= 0.007 and p= 0.043, respectively, Tukey HSD; Appendix B). 222 Expressed in percent consumption, 32.2% of Ceratodictyon spongiosum and 24.87% of H. 223 macroloba was consumed. Padina sp. was an intermediate food choice where 17.2% was 224 consumed, while the least preferred feed was E. acoroides where only 1.13% was consumed 225 (Fig. 4). The two most preferred food items, C. spongiosum and H. macroloba, were then used in 226 the no-choice feeding experiment. 227 40

35

30

25

) consumption in consumption ) 20 n=5 16.5 hrs 16.5 15

10

5 Mean % (±sd, % Mean 0 Padina sp. Ceratodictyon Halimeda macroloba Enhalus acoroides spongiosum

Experimental Feeds Control (+) Feeds 228 229 Figure 4. Mean percent consumption in D. setosum over 16. 5 hours when offered a choice of 230 four feed types (n= 5). Vertical lines indicate standard deviations. 231

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27733v1 | CC BY 4.0 Open Access | rec: 15 May 2019, publ: 15 May 2019 232 233 No-choice feeding experiment 234 The average consumption rate of H. macroloba was slightly higher (0.22 ± 0.17 g·echinoid-1hr-1) 235 than that of C. spongiosum (0.15 ± 0.05 g·echinoid-1hr-1) (Fig. 5; Appendix C). Non-parametric 236 Mann-Whitney U test showed that the consumption rate of the two preferred feeds in no-choice 237 feeding experiment did not show significant differences (p > 0.05; Appendix D). This indicates 238 that preferred feeds cannot be further ranked. 239

0.45

1 1 - 0.4

0.35

echinoid ⸱ 0.3

0.25

0.2

± hrs 48 in3) n= SD, 0.15 -1

hr 0.1 ⸱

0.05 Mean consumption rateS (g rateS consumption Mean 0 Ceratodictyon spongiosum Halimeda macroloba

Experimental Feeds Control (+) Feeds 240 241 Figure 5. Mean consumption rates (g⸱echinoid-1 hr-1 ± SD) by D. setosum in no-choice feeding 242 experiments after 48 hrs (n= 3). Vertical lines indicate standard deviations. 243 244 Comparing the two experiments, C. spongiosum was consumed less by D. setosum during 245 the no-choice feeding experiment compared to when it was offered with choices. On the other 246 hand, the echinoid consumes more of the H. macroloba when the urchins were not offered 247 choices in their feeds. 248 However, after the experiment, there were observable effects on the physical appearance 249 of the echinoids. Two of the three experimental echinoids fed in the C. spongiosum setup were 250 observed to have spine loss and died at the end of 48 hours (Fig. 6). It is noteworthy that the 251 consumption rate of C. spongiosum in the experimental setups did not substantially differ from 252 the weight lost from the control setup (blue bar in fig. 5). 253

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27733v1 | CC BY 4.0 Open Access | rec: 15 May 2019, publ: 15 May 2019 R2 R3 254 255 Figure 6. Observable spine loss and mortality of Diadema setosum in two replicates (R2 and R3) 256 in no-choice feeding experiment after 48 hours. 257 258 Discussion 259 260 Choice feeding experiment 261 The feeding choice experiment showed that D. setosum avoided E. acoroides and preferred C. 262 spongiosum and H. macroloba the most. Note the most preferred feed, the red alga C. 263 spongiosum, is in a symbiotic relationship with the sponge Haliclona cymaeformis where the 264 alga makes up the bulk of the organism while the sponge provides the structure to give it shape 265 and form, thus contributing to the formation of tiny holes (Azzini et al., 2008). It is possible that 266 the secondary metabolites of C. spongiosum is an optimal nutritional requirement for the 267 echinoid when fed in a short time. 268 Halimeda macroloba was also preferred by Diadema setosum in this experiment. The 269 result was contrary to the report of Coppard & Campbell (2007) in Hawaii, where H. macroloba 270 was readily avoided by D. setosum in grazing preference trials. This suggests that D. setosum in 271 different geographic locations may exhibit different preferences. Another contrary report by 272 Solandt & Campbell (2001) showed that the congeneric green macroalga H. opuntia was also 273 avoided by D. antillarum in feeding choice experiments, probably because of its secondary 274 metabolites and high calcareous content (>90%). But for D. setosum, these deterrent factors may 275 have an opposite effect making them feed more on its thalli. The most likely explanation is that 276 the calcium carbonate content of Halimeda varies between species. Another possible reason is 277 that most of the echinoids have a neutral gut pH and regularly ingest some carbonate material 278 (Pennings & Svedberg, 1993). This means the calcareous content of H. macroloba might not 279 have deterred the grazing activity of D. setosum. This also shows that different species of 280 Diadema may prefer different feeds, as studied by Coppard & Campbell (2007), where D. 281 savignyi and D. setosum, exhibit different feeding preferences. 282 Padina sp. was intermediately preferred by D. setosum. This corresponds to the findings 283 of Coppard & Campbell (2007) where P. pavonica was consumed and digested with ease by D. 284 setosum because of its relatively soft texture and low amounts of tannins and phenols. 285 By contrast, the low preference of D. setosum for E. acoroides may be due to its tough 286 texture although it has relatively high protein content (Moore et al., 2019). The recent study by 287 Moore et al. (2019) on D. setosum provided a similar result wherein seagrasses were less 288 preferred than seaweeds. However, of all the seagrasses found around Barranglompo Island, 289 Indonesia, E. acoroides was consistently preferred by D. setosum. Therefore, the general trend of 290 feeding preference by the echinoid was consistent with the results obtained in this study.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27733v1 | CC BY 4.0 Open Access | rec: 15 May 2019, publ: 15 May 2019 291 292 No-choice feeding experiment 293 There was no significant difference in the consumption rate of feeds eaten by D. setosum during 294 the 48 hours no-choice feeding experiments. 295 According to Solandt & Campbell (2001), the high calcareous content of Halimeda sp. 296 may be essential for the urchin to survive for extended periods. And they added that the high 297 carbonate content of the genus Halimeda caused the echinoid to consume greater biomass to gain 298 the required energy. However, since the current experiment lasted only for 48 hours, the 299 detrimental effect of secondary metabolites of H. macroloba may have not been enough to 300 inhibit any significant feeding. Duffy & Paul (1992) found that algal consumption rates were 301 influenced by nutritional quality and relative calcification levels, more than by secondary 302 metabolite content. This would appear to be a possible case of this feeding experiment. 303 Ceratodictyon spongiosum was less consumed despite being a preferred feed in choice 304 experiments. The same pattern of results was seen in the study of Seymour et al. (2013), where 305 food items preferred during choice feeding experiments were not highly consumed in no-choice 306 experiments. This demonstrates that no-choice consumption rates do not necessarily reflect 307 preferences. It is noteworthy that D. setosum fed on C. spongiosum alone showed obvious signs 308 of stress (spine loss) and mortalities after 48 hours. It was also mentioned that C. spongiosum 309 forms a symbiosis with a sponge. Since marine sponges degrade quickly in laboratory if not 310 provided with favorable conditions, it may have greatly affected the urchins. This feed therefore 311 probably had damaging effects that lead to mortality of the urchins due to water fouling. Since 312 the poor water quality in the C. spongiosum experimental setups had an adverse effect on the 313 experimental animals, the results of no-choice experiment are not conclusive. 314 The study was only limited to using aerated seawater and not a flow-through water system. 315 Therefore, it cannot accurately simulate the underwater conditions. The study was also limited to 316 using only the most common marine plant species found in TINMR, so not all plant species in 317 the area were selected and tested. 318 319 Conclusions 320 Diadema setosum exhibited differential feeding preferences on the four dominant marine plants 321 in TINMR. The percent consumption of the echinoid on different feeds varies significantly when 322 offered with choices. The sponge-symbiotic red alga C. spongiosum and the green alga H. 323 macroloba were the preferred species, and the seagrass E. acoroides was barely consumed. It can 324 be inferred that D. setosum forages more on macroalgae than seagrass species. However, there 325 was no difference on the consumption rate of D. setosum when offered with single-diet preferred 326 feeds, although this may be due to water quality problems with the experimental setup. The study 327 tends to support the ecological role of D. setosum as an important herbivore that regulates certain 328 macroalgal species in TINMR through its grazing activities. This algal grazing activity, in turn, 329 can promote scleractinian coral recruitment. 330 The use of flow-through seawater system in the laboratory for feeding experiments 331 should be used to have a better simulation of underwater conditions. Cage-field experiments can 332 be another alternative method to directly observe the feeding behavior of D. setosum on various 333 species of macroalgae. 334 To improve the determination of feeding preferences of D. setosum, more food choices 335 that are present in the forereefs of TINMR can be offered to the echinoid. The feeding preference 336 experiments of the echinoid can be conducted across a year (during wet and dry seasons)

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27733v1 | CC BY 4.0 Open Access | rec: 15 May 2019, publ: 15 May 2019 337 utilizing available feeds in TINMR to evaluate whether there are differences in the preferences of 338 D. setosum in different times of the year. Comparison of feeding preferences of different 339 Diadema species in TINMR can be conducted to establish similarities or differences in 340 terms of feeding ecology of the echinoids. Further experiments can be conducted to determine 341 factors (such as nutrients, palatability, and morphological features) that influence preferences of 342 D. setosum on the species it likes to graze on. 343 Gut content analysis can be incorporated in the methods in order to have a better view on 344 the type of food material the urchin feeds on. Finally, the development of feeds from the 345 preferred plant species of D. setosum can be done to increase gonad production which can help 346 aquaculture industry. 347 348 349 Acknowledgements 350 This work was partially supported by a University of the Philippines Balik-PhD grant to MCD 351 Malay. The authors would like to thank Protected Area Management Board (PAMB) of TINMR 352 and DENR Region VI, for granting them the permission to conduct this study. The first author 353 would personally like to thank the staffs in UPV- Marine Biological Station (MBS) at Taklong 354 Island for field sampling support, and Mr. Joseph Arbizo for the statistical analysis advice. 355 356 References 357 Alves F, Chicharo L, Serrao E, Abreu A. 2003. Grazing by upon algal 358 communities in rocky substrates. Scientia Marina 67 (3): 307-311. 359 Azzini F, Calcinai B, Cerrano C, Pansini M. 2008. Symbiotic associations of sponges and 360 macroalgae: the case of Haliclona cymaeformis and Ceratodictyon spongiosum from the 361 Ha Long Bay (Vietnam). Biologia Marina Mediterranea 15 (1): 248-249. 362 Cabanillas-Terán N, Loor-Andrade P, Rodriguez-Barreras R, Cortes J. 2016. Trophic ecology of 363 sea urchins in coral-rocky reef systems, Ecuador. PeerJ 4: e1578 [about 21 p.]. 364 Coppard SE, Campbell AC. 2007. Grazing preferences of diadematid echinoids in Fiji. Aquatic 365 Botany 86: 204-212. 366 Duffy JE, Paul VJ. 1992. Prey nutritional quality and the effectiveness of chemical defenses 367 against tropical reef fishes. Oecologia 90(3): 333-339. 368 Erickson A, Paul V, Van Alstyne K, Kwiatkowski L. 2006. Palatability of macroalgae that use 369 different types of chemical defenses. Journal of Chemical Ecology 32: 1883-1895. 370 Goh B, Lim D. 2015. Distribution and abundance of sea urchins in Singapore reefs and their 371 potential ecological impacts on macroalgae and coral communities. Ocean Science 372 Journal 50(2): 211-219. 373 Hay ME, Lee R Jr., Guieb R. 1986. Food preference and chemotaxis in the sea urchin Arbacia 374 punctulata (Lamarck) Philippi. Journal of Experimental Marine Biology and Ecology 96: 375 147-153. 376 Kasim M. 2009. Grazing activity of the sea urchin Tripneustes gratilla in tropical seagrass beds 377 of Buton Island, Southeast Sulawesi, Indonesia. Journal of Coastal Development 13(1): 378 19-27. 379 Kriegsch N, Reeves S, Johnson C, Ling S. 2016. Phase- shift dynamics of sea urchin overgrazing 380 on nitrified reefs. PLoS ONE 11(12): e0168333 [about 15 p.].

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PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27733v1 | CC BY 4.0 Open Access | rec: 15 May 2019, publ: 15 May 2019